Intracellular calcium (Ca2+) is one of the most important signaling molecules in cells, especially in neural tissues. Most effects of Ca2+ are transduced through specific Ca2+ binding proteins such as calmodulin (CaM) which interact with many proteins including a family of CaM- kinases (CaM-Ks). My laboratory has characterized several members of this CaM-K family in terms of molecular properties and regulation. More recently we have begin to investigate physiological functions for these CaM-Ks and identify their substrates. In this grant we will focus on the CaM-K cascade which is comprised of CaM-KK and its downstream targets CaM-KI, CaM-KI, CaM-KIV and protein kinase B (PKB). We will study the role of the CaM-K cascade in two important developmental aspects, apoptosis and neurite outgrowth, using cerebellar granule neurons. Aim 1. Role of CaM-K cascade in inhibiting apoptosis. A variety of dominant-interfering and constitutively active mutant of KK, KIV, PKB, CREB and CBP will be used to explore their roles in regulating apoptosis in granule cells maintained in either HK (25 mM) or LK (5 mM kci). Attempts will be made to identify regulated genes, such as those encoding BDNF or Bcl-2 family members. Aim 2. Role of CaM-K cascade in promoting neurite outgrowth. The same reagents as in Aim 1 will be employed to determine their effects on neurite outgrowth in granule neurons maintained in HK. Attempts will be to make to determine whether the CaM-K cascade is acting through nuclear events or at the dendrites and growth cone and to identify possible substrates of KI. These studies will further our understanding of the molecular pathways regulating these important developmental functions in neurons. They will also contribute to our knowledge of the physiological functions of the CaM-K cascade.